Wednesday, December 4, 2024
12/4/2024
PROJECT SUMMARY Malaria remains a global health disease that affects 40% of the world’s population and killed 619,000 in 2021. The etiologic agent of malaria are Plasmodium spp. parasites, of which Plasmodium falciparum is the most prevalent and deadly species. The World Health Organization recommends artemisinin-based combination therapies (ACTs) as first-line treatment for falciparum malaria, and dihydroartemisinin (DHA) is the active metabolite of all clinically-used artemisinins. Artemisinin resistance (ART-R) has been documented globally and is prevalent in the Greater Mekong Subregion of Southeast Asia, presenting a major hurdle to malaria eradication. The most well-characterized genetic marker of ART-R is mutations in the propeller domains of Kelch13, a protein that structurally resembles an E3 ubiquitin ligase adaptor. Our studies utilize isogenic parasites of Cambodian origin with mutations in Kelch13 and the proteasome. We have shown that P. falciparum-specific proteasome inhibitors kill ART-R parasites, and that mutations in the proteasome increase susceptibility to DHA. Moreover, proteasome inhibitors synergize with distinct classes of antimalarials that perturb proteostasis, including DHA. We and others have shown that DHA non-specifically and promiscuously alkylates heme and nearby parasite proteins. However, whether alkylated proteins are ubiquitinated and subsequently targeted to the proteasome for degradation remains unknown. In addition, it is unknown if these alkylated products are responsible for the parasite proteasome inhibition observed with DHA. Proteasome inhibition will lead to buildup of proteins that activate the unfolded protein response (UPR). In this regard, we observe that early parasite responses dictate eventual outcomes. Artemisinin-sensitive (ART-S) parasites demonstrate hyperactivation of the UPR at early ring stages and a subsequent inability to recover from UPR activation. Collectively, these data led us to hypothesize that a functional ubiquitin proteasome system (UPS) is necessary for ART-R. In Aim 1, we examine the role of the proteasome in ART-R. We will determine whether alkylated proteins are ubiquitinated and whether DHA-mediated alkylation inhibits the proteasome. In Aim 2, we examine the role of ubiquitination in ART-R. We will identify and interrogate the role of distinct ubiquitin branch patterns in conferring artemisinin survival. In Aim 3, we will determine if dysregulation of the UPS and UPR are generalities underlying artemisinin susceptibility. We will examine African parasites that are isogenic for mutations in coronin, AP2µ, and UBP1, proteins involved in endocytosis and ubiquitination that have been confirmed by gene editing to mediate ART-R, thus extending the implications of our earlier findings. An understanding of the molecular underpinnings of DHA and proteasome inhibitor synergy could extend the clinical utility of artemisinins, support proteasome inhibitor-based combination therapies, and lead to development of other antimalarials that perturb parasite proteostasis. The proposal aims to address knowledge gaps in parasite proteostasis with the intent of leveraging this knowledge for development of therapeutics to combat ART-R malaria.
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